Doctor of Philosophy
Microbiology and Immunology
Atherosclerosis is a chronic inflammatory disease characterized by thickening of the arterial wall from accumulation of lipoproteins and immune cells within the vessel intima, forming a lipid-rich plaque. Macrophages play a central role in atherosclerosis progression through phagocytic removal of apoptotic cells in a process termed efferocytosis. With disease progression however, macrophage efferocytosis becomes defective and uncleared apoptotic cells undergo secondary necrosis and form a necrotic core that increases the risk of plaque rupture. Despite the importance of defective efferocytosis in atherosclerosis, little is known about how efferocytosis becomes impaired. The aim of this thesis was to characterize mechanisms regulating efferocytic clearance of apoptotic cells and to identify potential mechanisms that contribute to impaired efferocytosis in atherosclerosis. Using a combination of mass spectrometry and microscopy-based efferocytosis uptake assays, we identified Rab17 as a regulator of apoptotic cargo trafficking to the recycling endosomes. Disruption of Rab17 function using a dominant-negative mutant resulted in mis-trafficking of apoptotic cargo away from recycling endosomes and into the MHC class II loading compartment. We then examined the gene expression profile of macrophages from the aortas of patients undergoing open heart surgery to identify potential mechanisms driving defective efferocytosis. We found over 3,000 differentially-expressed protein-coding genes in these macrophages, with particular enrichment in pathways involved in cholesterol handling, efferocytosis and efferosome maturation. Interestingly, we observed upregulation of the hematopoietic transcription factor GATA2, which has been shown by genetic linkage studies to be associated with coronary artery disease. By perturbing GATA2 expression in oxLDL-treated THP-1 human macrophages, we found that oxLDL-induced impairment in efferocytosis and efferosome maturation is dependent on GATA2. Our findings indicate that exposure to pro-atherogenic conditions induces upregulation of GATA2 expression, which specifically impairs efferocytic uptake of apoptotic cells and efferosome maturation. Our data shed new light on the process of macrophage efferosome maturation and how this process is impaired in atherosclerosis. We are also the first to describe a role for GATA2 in mediating oxLDL-induced impairment in efferocytosis. Therefore, GATA2 and other processes involved in dysregulation of efferosome maturation may be suitable therapeutic targets in human atherosclerotic disease.
Summary for Lay Audience
Atherosclerosis is a disease of the blood vessels involving the accumulation of cholesterol and cells of the immune system within the vessel wall. This results in the formation of an atherosclerotic plaque, which grows over time and may eventually rupture and cause a number of complications, including heart attack and stroke. A type of immune cell called macrophages can migrate into the atherosclerotic plaque and remove dying cells and debris from within the plaque in a process called ‘efferocytosis’. Macrophage efferocytosis helps limit the growth of the plaque. However, over time efferocytosis within the plaque becomes defective, and macrophages are no longer able to limit plaque growth leading to faster disease progression. In this thesis, we studied macrophages isolated from atherosclerotic plaques in human patients to understand how efferocytosis becomes defective in atherosclerosis. We first identified a new regulator of efferocytosis called Rab17, which helps the macrophage properly break down the dead cells and debris it takes up during efferocytosis. We then found that certain genes and pathways related to efferocytosis were dysregulated in the macrophages from patients. These included pathways related to how the macrophage processes excess cholesterol and how macrophages deal with the dead cells they take up. Finally, we identified a transcription factor (protein that regulates gene expression) that was associated with defective efferocytosis called GATA2. We found that an increase in the amount of GATA2 in the cell seemed to be required for macrophages to develop defective efferocytosis. Taken together, these results advance our understanding of atherosclerosis and uncovers a role for GATA2 in the development of atherosclerotic disease.
Yin, Charles, "The Role of Defective Macrophage Efferocytosis During Early Atherosclerosis in Humans" (2019). Electronic Thesis and Dissertation Repository. 6428.
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Available for download on Wednesday, January 01, 2020